Hepatitis B virus (HBV) is prevalent throughout the world, but is especially endemic to parts of Asia and Africa. Acute infection is usually resolved after 4-6 months; however, chronic infection often lasts a lifetime and is highly associated, inter alia, with the development of cirrhosis and primary hepatocellular carcinoma. See, for example, Beasley and Hwang, "Epidimiology of Hepatocellular Carcinoma," Chapter 16, pp. 209-224 in Vyas, Girish N. et al., eds., Viral Hepatitis and Liver Disease, (Grune and Stratton, Inc., Orlando, Fla., 1984), where it is reported that, on a worldwide basis, 80 percent of the people in whom primary hepatocellular carcinoma occurs are hepatitis B surface antigen (HBsAg) carriers. The 200 million chronic HBV carriers worldwide can act as a reservoir of infectious HBV in the general population. Accordingly, the development of an effective treatment of hepatitis B viral infections, especially of chronic hepatitis, is urgently needed to arrest the increasing spread of this disease.
Chronic HBV infection can be categorized into chronic active hepatitis (CAH) and chronic persistent hepatitis. CAH symptoms encompass fatigue, nausea, anorexia and/or abdominal pain, and general depression of liver function. Chronic persistent HBV infection is associated with cirrhosis of the liver. As noted above, acute HBV infection is usually resolved in 4-6 months. Its symptoms encompass fever, malaise, fatigue, anorexia, nausea, vomiting and abdominal pain. A small portion of acute HBV infections result in massive hepatic necrosis, coma, and death. No chemotherapeutic agent has yet been found to be effective for treatment of acute viral hepatitis (Vyas, Girish N. et al., eds., above, Chapter 8, pp. 115-122, at page 115).
Ideally, antiviral agents for HBV infection could be used to eliminate HBV infection from chronic carriers, thus decreasing their changes of developing HBV associated cirrhosis and primary heptocellular carcinoma. In addition, antiviral agents could be used to speed up recovery from acute HBV infection. At present no such chemotherapeutic agent has been identified. The development of such antiviral agents has been hindered by three major factors. First, the host range of HBV appears to be confined to humans, the natural host, and a few higher primates such as chimpanzees. Although three related viruses which infect the Beechey ground squirrel (Ground Squirrel Hepatitis Virus GSHV), the woodchuck (Woodchuck Heptatitis Virus: WHV) and the domestic duck (Duck Heptatitis B Virus: DHBV), have been found, none of these viruses can infect nor replicate in common laboratory animals such as rats and mice (Summers, Hepatology 1: 179-183, 1980). Secondly, no laboratories have been able to infect and propagate HBV in tissue culture. Recently, Tuttleman et al (Molecular Biology of Hepatitis B Viruses, Cold Spring Harbor Labs, pg. 9, 1985) have described a tissue culture system in which 5% primary duck hepatocytes can be infected with the duck hepatitis B virus (DHBV), a member of the hepatitis B virus family. However, since the protocol utilizes normal hepatocytes from young ducklings, the system cannot be readily adapted for propagation of HBV in human hepatocytes (i.e., lack of donor hepatocytes). Thirdly, the study of the mechanism of replication of HBV, characterization of the viral gene products, and elucidation of factors which influence the outcome of the infection has progressed slowly due to the aforementioned factors.
In spite of these obstacles, there have been attempts at and partial success in providing a method of treatment of hepatitis B virus by chemotherapy and/or immunotherapy. However, a practical, effective treatment for hepatitis B virus infection has not been found. One such attempt and partial success is described by C. I. Smith and William S. Robinson, et al., "Acute Dane Particle Suppression with Recombinant Leukocyte A Interferon in Chronic Hepatitis B Virus Infection," The Journal of Infectious Diseases, 148, 907-13 (1983). In this method Robinson and his coworkers used a leukocyte A Interfereon (rINF-A or HuIFN-2), produced by recombinant DNA methods, to treat nine patients with chronic hepatitis B virus infection. They report that most courses of rINF-A treatment were associated with a reduction in polymerase activity of the viron or Dane particle. However, this change was not permanent in any of the patients.
One means of developing an effective treatment for HBV infection is to invent a compound which inhibits the function of a particular viral protein which is necessary for viral replication, such as a viral-associated polymerase. Robinson and colleagues discovered a polymerase inside the HBV particles isolated from serum (Kaplan, et al., J. Virol 12: 995-1005, 1973). Its presence can be readily detected by the endogenous polymerase assay which measures the ability of the polymerase to incorporate radiolabeled nucleotides into the viral genome. Further studies showed that only one strand, called the short strand or +strand was being synthesized in each particle (Summers et al, Proc. Nat'l. Acad. Science 72, 4597-4601, 1975; Landers et al., J. Virol. 23, 368-376, 1977). Using the related duck hepatitis B virus animal model, Summers and Mason (Cell 29, 403-413, 1982) have isolated immature viral particles from infected duck hepatocytes and showed that (1) the RNA pregenome was packaged in immature particles, (2) an endogenous polymerase synthesized the minus strand DNA from the RNA template by reverse transcription (3) the RNA template was degraded by an RNAse H activity present in the particles, (4) the plus strand was synthesized using the minus (-) strand DNA as a template, and (5) the polymerase associated with the immature viral particles did exhibit reverse transcriptase-like activity. Recent studies of HBV, GSHV, and WHV DNA intermediates isolated from infected liver revealed asymmetric synthesis of minus strand, consistent with the above findings (Weiser et al., J. Virol. 48, 1-9, 1983; Fowler et al., J. Med. Virol. 13, 83-91, 1984; Roggendorf and Summers, Molecular Biology of Hepatitis B Viruses, Cold Spring Harbor Laboratories, pg. 5, 1985). Although no laboratory has directly shown that the polymerase found in the viral particles is encoded by the virus itself, Toh et al (Nature 305, 827-829, 1983) have reported the existence of extensive amino acid sequence homology in specific regions of a large open reading frame encoded by HBV (adr strain) and WHV and three other retroviral reverse transcriptases. This analysis lends some credence to the hypothesis that the reverse transcriptase associated with viral particles is encoded by the viral genome.
Since the HBV polymerase exhibits reverse transcriptase properties, one means of arriving at a method of treatment would be to test known inhibitors of similar reverse transcriptases, for example retroviral reverse transcriptases. Some rifamycin derivatives have been found to inhibit particular reverse transcriptases. The following articles disclose certain rifamycin derivatives which inhibit particular reverse transcriptases (polymerases): Maurice Green et al., "3 Cyclic Amine Derivatives of Rifamycin: Strong Inhibitors of the DNA Polymerase Activity of RNA Tumor Viruses," Proceedings of the National Academy of Sciences (P.N.A.S.), 69, 1294-98 (1972); Frances M. Thompson et al., "Inhibition of Three Nucleotide Polymerases by Rifamycin Derivatives," P.N.A.S. 71, 107-9 (1974); Alan M. Wu, et al., "RNA Directed DNA Polymerase and Virus-Induced Leukemia in Mice," P.N.A.S. 70, 1298-1302 (1973); and Corrado Gurgo et al., "Rifamycin Derivatives strongly Inhibiting RNA DNA Polymerase (Reverse Transcriptase) of Murine Sarcoma Viruses," Journal of the National Cancer Institute, 49, 61-79 (1972).
Applicant herein has now surprisingly discovered a few rifamycin derivatives which inhibit the polymerase activity of hepatitis B virus. Such discovery provides means for treating hepatitis brought on by infection with hepatitis B virus.